Proteomic analysis
The proteomic analysis of the leaf tissues including protein extraction, two-dimensional electrophoresis (2-DE) separation, gel analysis and MALDI-TOF/TOF analysis were performed according to our previous reports.18
Briefly, total leaf proteins were extracted by phenol method, and quantified by a 2-D Quant Kit (GE Healthcare Amersham Bioscience) according to the manufacturer’s instructions. Then a 2-DE process was performed for protein separation and 2-DE gel map acquisition. The protein suspended in the lysis buffer (8 M urea, 2 M thiourea, 4 % CHAPS, 1 % DTT, 1 % IPG buffer pH 4–7) was loaded to strips (Immobiline Dry Strip, pH 4-7,18 cm; GE Healthcare) by overnight rehydration. Then the stripes were transferred into an Ettan IPGphor system (GE Healthcare Amersham Bioscience) for isoelectric focusing (IEF). After the IEF, reduction and alkylation reaction were carried out sequentially. For the second dimension electrophoresis separation, 12.5% SDS polyacrylamide gels were prepared. The strips were placed on top of the prepared SDS-PAGE gels and electrophoresis was implemented in an electrophoresis system (Bio-Rad).
The obtained 2-DE gels underwent a staining procedure with Coomassie Brilliant Blue R-250, and were scanned by an image scanner at a resolution of 600 dpi. PDQuest software (Version 8.01, Bio-Rad, United States) were used for the gels analysis and the screening for protein spots showing changed intensity among the gels. The threshold for the changed protein spots: intensity variation no less than twofold, Student’s t-test with p < 0.05.
The significant changed protein spots were excised manually from 2-DE gels, trypsin digestion, peptide extraction and vacuum drying were performed sequentially. The resulted peptides were mixed with saturated matrix solution (saturated α-cyano-4-hydroxycinnamic acid in 50% ACN/0.1% TFA) and were in order spotted on a stainless steel target plate for MS analyses.
The MALDI-TOF-TOF mass spectrometer (AB SCIEX TOF/TOF, 5800 system) was used for protein identification. The parameters and procedures for MS acquisition and database searching were performed referring previous report.18 Briefly, positive scan mode was selected and mass range was set as 850-4000 Da for MS1. Five precursors were selected for MS2 analysis. The MS data were searched against the National Center for Biotechnology Information non-redundant (NCBInr) database, and the taxonomy of green plants was selected using Mascot search engine (http://www.matrixscience.com). The search parameters were listed as follows: Trypsin/P was specified as the cleavage enzyme and one missed trypsin cleavage was allowed. Fixed modification was defined as carbamidomethylation on cysteine and variable modification was defined as oxidation on methionine. Mass errors for precursors and fragments were 100 ppm and 0.3 Da, respectively. The false discovery rate was defined as 1% for positive identification. All the rest parameters followed the default settings in Mascot.